Mechanistic insights into recovery of partial denitrification/anammox under continuous flow: Balancing nitrite supply...

Partial denitrification coupled with anammox (PD/A) has emerged as a promising low-carbon strategy for energy-efficient nitrogen removal from municipal wastewater. However, the reactivation of PD/A systems following operational disturbances remains challenging, particularly under continuous-flow conditions, where microbial interactions and process stability are more complex than in sequencing batch reactors. This study systematically and first evaluated the recovery dynamics of a continuous-flow PD/A process seeded with low-activity granular sludge stored at 4 °C for three months. The recovery was conducted through seven operational phases, targeting sequential restoration of partial denitrification and anammox activities via modulation of influent nitrate concentrations, carbon source dosing patterns, and bioaugmentation. Denitrifying bacteria exhibited faster reactivation than anammox bacteria, resulting in insufficient nitrite (NO₂^-) accumulation for sustained anammox activity. Increasing influent NO₃^--N to 80 mg/L improved the NO₃^--to-NO₂^- transformation ratio (NTR) to 49.1 %, yet failed to meet NO₂^- demands. Intermittent carbon dosing further elevated NTR to 87.4 %, although the specific NO₂^- reduction rate remained suboptimal (13.5 mg N/g VSS/h). Direct supplementation of NH₄⁺ and NO₂^- for anammox recovery yielded a nitrogen removal efficiency (NRE) of only 0.7 %, indicating that nitrite availability-not merely presence-governs system recovery. Bioaugmentation with fresh PD/A sludge significantly accelerated reactivation, achieving a stable NRE of 88.2 %. Microbial community analysis revealed dynamic shifts in sludge morphology and dominant populations during recovery of PD/A process. Granular sludge supported the retention of anammox bacteria but also enriched Chloroflexi (41 %), which was regarded as competitor with denitrifiers for carbon sources. Meanwhile, flocculent sludge dominated by Zoogloea (40.9 %) emerged during instability, contributing to extracellular polymeric substance production and system heterogeneity. This study provides mechanistic insight into microbial competition and process limitations during PD/A system recovery under continuous-flow conditions, offering practical guidance for robust implementation in full-scale applications.